PCID2 influences BRCA1/BARD1 Localization and Centrosome Duplication through its functions in Nuclear Protein and mRNA Export

Abstract

Nuclear protein export has an emerging role in the regulation of centrosome duplication, a function that involves interaction of centrosomal proteins with the Crm1 nuclear export factor. The protein PCID2, while originally identified as an mRNA export factor, is also known to function in Crm1 mediated protein export, with PCID2 siRNA knockdown slowing the rate of export. In addition, PCID2 has been localized to the centrosome. Despite the fact that PCID2 is poised to be a cell cycle regulator through potential combination of it's centrosomal and nuclear export roles, the function of PCID2 at these locations has not been well characterized. We sought to elucidate a potential link between these roles of by exploring PCID2's involvement in the export of particular cargo known to transit between these the nucleus and centrosome. Our studies focus on BRCA1 and BARD1 as potential PCID2 nuclear to centrosomal cargo, as these DNA damage repair proteins transit from the nucleus to the centrosome in a Crm1 dependent manner, particularly in times of cellular stress. We found that knockdown of PCID2 caused a 10% increase in the nuclear retention of BRCA1, with a concomitant 40‐fold decrease in centrosomes exhibiting BRCA1 localization. These results suggest a role for PCID2 in the export of the BRCA1 protein from the nucleus and in it's subsequent localization to the centrosome. Conversely, BARD1 nuclear levels decreased by nearly 20% in the absence of PCID2, which was accompanied by a 40‐fold decrease in BARD1 positive centrosomes. These effects appear to stem from a 50% drop in BARD1 protein levels in PCID2 knockdown cells, suggesting a potential role for PCID2 in the export of BARD1 mRNA rather than protein, with failure to export contributing to loss in protein expression. Additionally, we discovered that altered transport of BRCA1/BARD1 due to PCID2 knockdown had an unexpected effect on centrosome duplication. Rather than the increase in centrosome number seen in the absence of the BRCA1/BARD1 interacting protein OLA1, PCID2 knockdown led to a four‐fold reduction in cells with an aberrant number of centrosomes (3 or more) as visualized by yTubulin staining. This variation is potentially due to the use of non‐breast cancer cells in this work. Our ongoing studies seek to identify CRM1‐PCID2‐BRCA1 export complexes and determining the effect of PCID2 knockdown on the CRM1‐BRCA1 interaction. In addition, we are investigating the role of PCID2 in BARD1 mRNA export. Determining how PCID2 influences the cellular localization of the BRCA1/BARD1 complex will help to elucidate the mechanism of PCID2's effect on centrosome duplication and therefore determine it's potential impacts on cell cycle regulation and tumor development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.